柑橘不同倍性种质胚性愈伤组织诱导及无病毒植株再生  

Embryogenic callus induction and virus-free plantlet regeneration from seedless cultivars in citruses of different ploidy

作　　者：任家勤 黄雪冰 杨雯惠 陈昊 邓家锐 敖义俊 解凯东[1] 郭文武[1] 伍小萌 REN Jiaqin;HUANG Xuebing;YANGWenhui;CHEN Hao;DENG Jiarui;AO Yijun;XIE Kaidong;GUOWenwu;WU Xiaomeng(National Key Laboratory for Germplasm Innovation&Utilization of Horticultural Crops,Wuhan 430070,Hubei,China;Chenggu Fruit Technology Guidance Station,Chenggu 723200,Shaanxi,China)

机构地区：[1]华中农业大学果蔬园艺作物种质创新与利用全国重点实验室,武汉430070 [2]城固县果业技术指导站,陕西城固723200

出　　处：《果树学报》2024年第7期1332-1341,共10页Journal of Fruit Science

基　　金：国家自然科学基金项目(32072528);湖北省自然科学基金项目(2022CFB157);湖北省重点研发计划项目(2022BBA0019);南宁市科技重大专项(20222061);国家柑橘产业技术体系项目(CARS-26)。

摘　　要：【目的】通过离体培养败育胚珠诱导柑橘二倍体和四倍体种质的胚性愈伤组织,为柑橘多倍体研究提供成对离体材料;继续诱导胚性愈伤组织再生无毒种苗实现柑橘无核品种提纯复壮。【方法】从柑橘成熟果实中挑取败育胚珠,接种于3种愈伤诱导培养基中,在离体培养过程中诱导并驯化胚性愈伤组织;再诱导愈伤组织分化为胚状体和不定芽,将不定芽嫁接到枳橙砧木形成完整植株;通过InDel标记和病毒检测鉴定再生植株的遗传来源和脱毒效果。【结果】离体培养诱导出13个柑橘种质的胚性愈伤组织,其中8个愈伤组织完成驯化,分别为红橘二倍体/四倍体(2x/4x)、W.默科特橘橙2x/4x、鄂柑1号椪柑2x/4x、日辉橘2x和早红脐橙。基因型影响柑橘胚性愈伤组织诱导效率,早红脐橙诱导率高达74.73%,而红橘诱导率仅6.85%;柑橘四倍体愈伤组织诱导率(0.56%~22.61%)低于对应二倍体(6.85%~37.75%)。3种不同培养基的胚性愈伤组织诱导效率不同,其中MGS培养基对温州蜜柑在内的5个品种的诱导率较高(14.13%~63.01%),MK培养基对温州蜜柑在内的4个品种的诱导率较低(21.62%~69.51%),但MK培养基对椪柑2x和红橘2x的诱导率分别为70.21%和17.31%。分别获得早红脐橙、伦晚脐橙、国庆1号温州蜜柑、大分4号温州蜜柑和兴津温州蜜柑5个无核品种的愈伤组织再生苗23、22、20、10和15株;嫁接嵌合体早红脐橙的愈伤组织再生苗经InDel标记鉴定实为罗伯逊脐橙;国庆1号温州蜜柑、伦晚脐橙和早红脐橙再生苗经PCR检测证明未感染黄脉病和衰退病病毒。【结论】柑橘胚性愈伤组织诱导率受品种基因型和培养基影响;同一品种的不同倍性种质,二倍体愈伤组织诱导率和胚状体发生率均较高;胚性愈伤组织再生植株实现了病毒完全脱除,是柑橘无核品种提纯复壮的有效手段。【Objective】Citrus is the biggest fruit industry in China and in the world.China is the origin center of citrus,owning abundant and diverse citrus germplasm resources,and it is important to properly conserve and utilize the elite citrus germplasm.The embryogenic callus induced from the aborted ovules is genetically identical to the original explant and capable of regeneration,making it feasible for in vitro conservation of citrus germplasm.The embryogenic callus also provides in vitro materials for studies of important traits,such as somatic embryogenesis,fruit quality control and stress resistance.We have induced and preserved embryogenic callus germplasm of over 100 different genotypes.However,the embryogenic calluses are all induced from the diploid germplasm,while induction of embryogenic callus from polyploid germplasm has been rarely reported.The polyploid is characterized by giant organ,dwarf plant and stronger adaptability.The polyploid embryogenic callus would accelerate basic research and applications of citrus polyploidy,by providing in vitro materials with short growth cycle,stable status,and easily accessibility.In addition,the whole new plantlets regenerated from embryogenic callus are always virus-free,which are promising in resolving the problem of fruit yield and quality delineation in seedless cultivars caused by virus infection after many years propagation by grafting.In this study,the aborted ovules of diploid and tetraploid citrus germplasm were cultured to induce embryogenic callus,to provide in vitro materials for citrus ploidy research.Regeneration of virus-free plantlets from embryogenic callus achieves purification and rejuvenation of seedless cultivars like navel orange and Satsuma mandarin.【Methods】The aborted ovules from mature fruits were inoculated on three types of callus induction mediums(MES:MT+ME 0.5 g·L-1+SAD 40 mg·L-1;MGS:MT+ME 0.5 g·L-1+SAD 40 mg·L-1+GA31 mg·L-1;MK:MT+KT 0.5 mg·L-1)under sterile conditions to induce and train embryogenic callus,and the ploidy of

关 键 词：柑橘 胚性愈伤组织 倍性种质 无核品种 离体再生 

分 类 号：S666[农业科学—果树学]